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Postnatal development and laminar distribution of noradrenergic fibers in cat visual cortex
DEVELOPMENTAL BRAIN RESEARCH Developmental Brain Research 82 (1994) 90-94
ELSEVIER
Research report
Postnatal development and laminar distribution of noradrenergic fibers in cat visual cortex Yulin Liu *, Max Cynader Department of Ophthalmology, University of British Columbia, 2550 Willow Street, Vancouver, BC, Canada V5Z 3N9
Accepted 24 May 1994
Abstract Previous studies have indicated that adrenergic receptors show significant changes either in laminar distribution or in number during the critical period of kitten visual cortex development. In order to further investigate the postnatal developrnent of this neurotransmitter system, especially in relation to the critical period, we used a polyclonal antibody against dopamine-/3-hydroxylase to localize noradrenaline-containing afferents in visual cortex of kittens of various ages from birth to adulthood. In young kittens, less than 2 weeks of age, noradrenergic fibers were sparse, short and randomly oriented, and were concentrated in layer I and in deep cortical layers V and VI. By postnatal day 40, the fibers were present throughout all cortical layers and exhibited higher densities in layers I, II, III, V and VI, with a band of lower staining in layer IV. While tangential fibers predominated in layers I, V and VI, relatively straight radial fibers traversed layers II and III. After postnatal day 40, we did not find major changes in the laminar distribution of adrenergic fibers. This developmental laminar distribution pattern of adrenergic fibers resembles that of the /3-adrenergic receptors that we and others have studied in kitten visual cortex, but differs from that of a-adrenergic receptors. Keywords: Plasticity; Dopamine-/3-hydroxylase; Development; Cat; Visual cortex
1. Introduction Cortical synaptic modification due to n e u r o n a l activity occurs mainly within a short period of postnatal life and not in adult visual cortex. [11,12]. This transient 'critical p e r i o d ' has b e e n observed in the visual cortex of several species, and previous studies have shown that m a n y factors may play important roles in regulating this visual cortex plasticity [3,5,10,14]. T h e r e is evidence that cortical infusion of 6-hydroxydopamine, which destroys c a t e c h o l a m i n e terminals and depletes cortical noradrenaline, can prevent the shift o f cortical ocular d o m i n a n c e that is p r o d u c e d by m o n o c u l a r deprivation during the critical period [14], although some m o r e recent evidence points to a m o r e complex picture [3,6-8]. In previous studies, we r e p o r t e d that a - and fl-adrenergic receptors r e a c h e d their p e a k concentration in visual cortex within the critical period of visual cortex plasticity [17]. In addition, the n u m b e r and
* Corresponding author. Fax: (1) (604) 875-4663. 0165-3806/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0165-3806(94)00100-E
laminar distribution of both a - and fl-adrenergic receptors were shown to be regulated by lateral geniculate nucleus input. In o r d e r to further explore the relationship between noradrenaline and visual cortex development, we used a polyclonal antibody against dopamine-/3-hydroxylase to localize noradrenalinecontaining afferents in developing cat visual cortex and c o m p a r e d this developmental pattern with that of various classes of n o r a d r e n e r g i c receptors.
2. Materials and methods 2.1. Surgery
A total of twenty cats (see Results) of various ages were used in this study. For surgery, kittens were deeply anesthetized with halothane to effect; adult cats were anesthetized with i.v. sodium thiopental. In four animals (two kittens and two adult cats), a 1 mm thick trench of cortical tissue was aspirated by suction to a depth of 1 cm from the cortical surface (extending down to the corpus callosum) and extending from the midline to about 7 mm lateral of the midline on one side of the brain. The section was located at about A.P. 8.0 anterior and had the effect of interrupting modulatory fibers
Y. Liu, M. Cynader/ Developmental Brain Research 82 (1994) 90-94 (cholinergic, adrenergic, serotonergic and histaminergic) arising from brainstem and basal forebrain sources.
a .,
2.Z Immunocytochemistry Kittens of various ages (see Results) were used to study the normal postnatal development of dopamine-fl-hydroxylase in the developing visual cortex. Animals were deeply anesthetized with sodium pentobarbital and perfused through the aorta with 1% sodium nitrite in 0.1 M phosphate buffer (PB, pH 7.4) for 1 min followed by 4% paraformaldehyde in PB (PFA). The brains were then postfixed in 4% PFA overnight at 4°C. Blocks containing visual cortex were dissected and cut into 50 g m sections on a vibratome. After washing in PB for 1 h, the sections were incubated with a polyclonal antibody against dopamine-/3-hydroxylase (1:1000) (Anti-DBH antibody, Chemicon International Inc) and 5% normal goat serum in PB at 4°C overnight. 2.5% Triton was used to increase the penetration of the antibody within the tissue. After another series of washes in PB (6× 10 min), the samples were incubated in a second antibody (anti-rabbit IgG), followed by a series of washes in PB and incubation in avidin-biotin-HRP (1/1000, Vector) at room temperature for 45 min. Sections were then rinsed again in PB (2 x 10 min), followed by 0.05 M Tris-HCl (pH 7.6) (3 × 10 min). The immunoreaction was visualized with 0.005% 3',3'-diaminobenzidine and 0.0015% H 2 0 z. Sections were then washed in PB, dehydrated in an ascending concentration of ethanol and xylene, then coverslipped with Permount. Special care was taken to treat tissues from animals of different ages in the same way. The quality of the immunoreaction was tested by omitting the primary antibody during the process and very low immunoreactivity was found in these sections. While the DBH immunoreactive cell bodies were mostly located in the locus coeruleus of the brain stem, as has been shown frequently, the immunoreactive axons were present throughout the brain [4,9,16,21, 23,24]. The highly selective labelling observed suggests specific staining with this antibody. In addition, the results of other studies in the brains of several species have been shown that this antibody is specific in recognizing DBH [4,9,16,21,23,24]. Adjacent sections were stained with Cresyl violet and were used to establish laminar borders in the various visual cortical areas based on cytoarchitectonic criteria established by Otsuka and Hassler in adult cats and Shatz and Luskin for early postnatal development [18,20]. The electrophysiologically defined maps of Tusa which describe the relationships of regional borders and gyral patterns also contributed to defining cortical areas and layers [22]. For the lesion animals, a camera lucida system was used to draw all dopamine-/3hydroxylase immunopositive fibers within a 2 mm wide zone in the coronal plane of visual cortex of both hemispheres. Fibers throughout the cortical thickness were drawn and quantitative analysis was carried out to count the number of fibers in both the lesion and unoperated hemisphere.
3. Results
Immunoreactive fibers were found throughout the brain, with high densities in brain stem, thalamus and cortex. In the locus coeruleus of the brain stem, a number of neurons, as well as their axons were heavily stained (data not shown). 3.1. Laminar distribution and morphology of DBH-immunoreactivity in developing cat visual cortex
Kittens of various ages (postnatal day 1, 10, 20, 30, 40, 60, 90, 120 and adult) were used to study the
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Fig. 1. Photomicrographs of dopamine-fl-hydroxylase immunoreactive processes in coronal sections of developing kitten visual cortex drawn by camera lucida. Immunoreactive axons were more dense in layers I, V and VI initially, and then in older animals in layers I-III, V and VI, with lower density in layer IV. a: postnatal day 10. b: postnatal day 20. c: postnatal day 40. d: adult. Bar = 300 /~m.
developmental pattern of laminar distribution of dopamine-/3-hydroxylase in the visual cortex. The immunoreactivity was present only in axons in the visual cortex and immunoreactive axons displayed a high density varicosities throughout postnatal development (Fig. 1-3). No immunoreactive dendrites and cell bodies were found within visual cortex. In neonatal kittens, only a few immunoreactive fibers were stained. They were most concentrated in deep cortical layers and layer I. These immunoreactive fibers were confirmed as axons using morphological criteria. While the axons in layer I were mostly tangentially oriented, those found in the deep cortical layers ran in a predominantly vertical direction. At postnatal days 10 and 20, many more immunoreactive fibers were found throughout the visual cortex. These fibers were still most dense in deep cortical layers V and VI and layer I. However, many more fibers could be found in superficial layers II and III compared with postnatal day 1. At postnatal day 40, the immunoreactive fibers were most dense in the superficial layers (layers I-III) and deep cortical layers. Virtually all the immunoreactive fibers exhibited varicosities along their length (Fig. 3). After postnatal day 40, the pattern of laminar distribution of immunoreactive fibers showed little further alteration. However, the apparent density of immunoreactive fibers continued to increase until postnatal day 120. After postnatal day 120, we observed no further change either in laminar distribution or in density. That is, fibers were most dense in deep cortical layers V and VI and superficial layers I-III, with a lower band of density in layer IV. 3.2. Interruption of noradrenaline innervation to visual cortex
The effects on dopamine-3-hydroxylase immunoreactivity in cat visual cortex after lesions of the dorsal noradrenergic bundle were observed in four animals.
92
E Liu, M. Cynader / Del~elopmental Brain Research 82 (1994) 90-~)4
Two kittens were operated at 11 days of age and were perfused at postnatal day 40. Two adult cats were operated and sacrificed after a 3 month survival. The surgery was restricted to one side of the brain. We carefully counted the number of DBH positive fibers in a series of 2 mm wide fields extending through the cortical thickness in all 4 animals. Fig. 4 shows that the number of DBH immunoreactive fibers was reduced by about 80% on the lesion side (Fig. 4). However, the laminar distribution pattern of the few DBH immunoreactive fibers on the lesion side still resembled that of normal pattern of adult cat.
4. Discussion
In this study, we have used a polyclonal antibody against dopamine-/3-hydroxylase to examine the development of noradrenergic fibers in kitten visual cortex. We also examined the effect of surgical interruption of neuromodulatory pathways on the density and laminar distribution of dopamine-/3-hydroxylase immunoreactive fibers within the visual cortex. In general, noradrenergic fibers were sparse, short and randomly oriented, and were concentrated in layer I and in deep cortical layers V and VI in neonatal kittens. By 40 days of age, the fibers were present throughout all cortical layers and exhibited higher densities in layers I, II, III, V and VI, with a band of lower staining in layer IV. This developmental pattern of laminar distribution of noradrenergic fibers strongly resembles that of the development of /3-adrenergic receptors, which display a higher density of binding in deep cortical layers right after birth and then a concentration in superficial and deep cortical layers after postnatal day 40 [17]. The relatively weak DBH immunoreactivity in cortical layer IV in either neonates or during the peak of critical period is somewhat unexpected and surprised, since another group of adrenergic receptors, namely a-l- and a-2-adrenergic receptors show a strong tendency for binding in this layer and in the subplate zone at these ages [13]. In addition, this group of adrenergic receptors can be regulated by visual input. The mismatch in the distribution and laminar developmental pattern may suggest that the a-adrenergic receptors are not likely to be presynaptic receptors in visual cortex as has been described in other systems. It may be that the a-adrenergic receptors receive their inputs in a paracrine fashion primarily with long-distance diffusion from the noradrenergic fibers of nearby layers or that the relatively small number of fibers in the layer IV result in a-adrenergic receptor activation preferentially. As one of the major modulatory neurotransmitters in cerebral cortex, noradrenaline has been reported to
play an important role in regulating cortical plasticity [3,14]. The involvement of noradrenaline in regulating visual cortical plasticity has been based on evidence that cortical interruption of noradrenalinc syslcm can prevent the normal ocular dominance shift produced by monocular deprivation in kittens [1,3,8,15]. These finding are controversial but it appears likely that noradrenaline plays at least some role in cortical plasticity [3]. In this study, we showed that immunoreactive dopamine-/3-hydroxylase increased progressively after birth and reached its peak of postnatal development during the most sensitive period of visual cortex plasticity. This result provides morphological evidence to support a role for noradrenaline in regulating visual cortex development. Surgical interruption of the dorsal noradrenergic bundle depleted about 80% of dopamine-/3-hydroxyl-
Fig. 2. Photomicrographs of dopamine-fl-hydroxylase immunoreactive processes in coronal sections of adult cat visual cortex. These were most dense in superficial layers I - I l l , intermediate in layers V and VI, with the lowest density in layer IV. Bar : 300 ~tm.
Y. Liu, M. Cynader / Developmental Brain Research 82 (1994) 90-94
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Fig. 3. High magnification view showing dopamine-/3-hydroxylase immunoreactive varicosities during postnatal development. 50 g m coronal sections were taken from kitten visual cortex, a: postnatal day 1. b: postnatal day 40. c: adult. Bar = 50/xm.
ase immunoreactive fibers, yet we have shown that this manipulation has very little effect on either a- or /3-adrenergic receptor density within kitten visual cortex. The lack of effect of this major loss of noradrenergic input is somewhat surprising, but it may be that the 20% of noradrenaline fibers that remain intact provide enough input to prevent the receptor regulation that might be expected to occur. In this light, it is interesting that Daw concluded that reduction of the noradrenaline content of the visual cortex by 70 to 90% is insufficient to prevent the ocular dominance shift that occurs in the visual cortex after monocular deprivation [7,8]. The distinctive varicosities that characterized dopamine-fl-hydroxylase immunoreactive axons can be visuNormal side
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Fig. 4. Camera lucida drawing of dopamine-/3-hydroxylase immunoreactive fibers in adult animals after dorsal noradrenergic bundle lesions. Left panel: normal hemisphere, dopamine-/3-hydroxylase immunoreactive fibers were most dense in superficial and deep cortical layers. Right panel: lesioned hemisphere. About 80% depletion of dopamine-fl-hydroxylase immunoreactive fibers was observed. The heaviest concentration of fibers was still found in superficial and deep cortical layers. Bar = 300/zm.
alized throughout all the cortical layers during postnatal development. The innervation of noradrenaline to rat visual cortex has been suggested to operate at least partly through non-junctional diffusion of transmitter [19], and this pattern of innervation would affect adjacent multiple elements including neurons and glia as well as blood vessels [2,17].
References [1] Allen, E.E., Blakemore, L.J., Trombley, P.Q. and Gordon, B., Timing of 6-hydroxydopamine administration influences its effects on visual cortical plasticity, Dev. Brain Res., 32 (1987) 53-58. [2] Aoki, C., Beta-adrenergic receptors: astrocytic localization in the adult visual cortex and their relation to catecholamine axon terminals as revealed by electron microscopic immunocytochemistry, J. Neurosci., 12 (1992) 781-792. [3] Bear, M.F. and Singer, W., Modulation of visual cortical plasticity by acetylcholine and noradrenaline, Nature, 320 (1986) 172176. [4] Chang, H.T., Noradrenergic innervation of the substantia innominata: A light and electron microscopic analysis of dopamine beta-hydroxylase immunoreactive elements in the rat, Exp. Neurol., 104 (1989) 101-112. [5] Cynader, M., Shaw, C., Prusky, G. and Van Huizen, F., Neural mechanisms underlying modifiability of reponse properties in developing cat visual cortex. In B. Cohen. and Bodis-Woller (Eds.), Vision and the Brain, Raven Press, New York, 1990, pp. 85-108. [6] Daw, N.W., Rader, R.K., Robertson, T.W. and Ariel, M., Effects of 6-hydroxydopamine on visual deprivation in the kitten striate cortex, J. Neurosci., 3 (1983) 907-914. [7] Daw, N.W., Robertson, T.W., Rader, R.K., Videen, T.O. and Coscia, C.J., Substantial reduction of cortical noradrenaline by lesions of adrenergic pathway does not prevent effects of monocular deprivation, J. Neurosci., 4 (1984) 1354-1360.
94
E Liu, M. Cynader / Det,elopmental Brain Research 82 (1994) 90- 94
[8] Daw, N.W., Videen, T.O., Rader, R.K. et all, Substantial reduction of noradrenaline in kitten visual cortex by intraventricular injections of 6-hydroxydopamine does not always prevent ocular dominance shifts after monocular deprivation, Exp. Brain Res.. 59 (1985) 30-35. [9] Goldstein, M., Studies on catecholamine-synthesizing enzymes, Neurochem. Int., 17 (1990) 291-295. [10] Gu, Q. and Singer, W., Involvment of serotonin in neuronal plasticity of kitten visual cortex, Abstr. IBRO, 1991. [11] Hubel, D. and Wiesel, T.N., Receptive fields of cells in striate cortex of very young, visually inexperienced kittens, Z NeurophysioL, 26 (1963) 994-1002. [12] Hubel, D. and Wiesel, T.N., The period of susceptibility to the physiological effects of unilateral eye closure in kittens, J. Physiol, 206 (1970) 419-436. [13] Jia, W., Liu, Y.L. and Cynader, M.S., Development and regulation of alpha adrenergic receptors in kitten visual cortex, in press. [14] Kasamatsu, T. and Pettigrew, G., Depletion of brain catecholamines: failure of ocular dominance shift after monocular occlusion in kittens, Science, 194 (1976) 206-209. [15] Kasamatsu, T., Pettigrew, J.D. and Ary, M., Cortical recovery from effects of monocular deprivation: acceleration with norepinephrine and suppression with 6-hydroxydopamine, J. NeurophysioL, 45 (1981) 254-266. [16] Kosofsky, B.E., Molliver, M.E., Morrison, J.H. and Foote, S.L, The serotonin and norepinephrine innervation of primary visual cortex in the cynomolgus monkey (Macaca fascicularis ), J. Comp. NeuroL, 230 (1984) 168-178. [17] Liu, Y., Jia, W.G., Strosberg, A.D. and Cynader, M., Development and regulation of /3-adrenergic receptors in kitten visual
[18]
[19]
[20]
[21]
[22]
[23]
[24]
cortex: an immunocytochemical and autoradiographic ~tudy. Brain Res., 632 (1993) 274 286. Otsuka, R. and Hassler, R., Uber Autbau and Gliederung der Corticalen Sehsphare bei der Katze, Arch. Psychiatr. Ncrrenkr., 203 (1962) 212-234. Ouimet, C.C., Patrick, R.L. and Ebner, F.F., An ultrastructural and biochemical analysis of norepinephrine-containing varicosities in the cerebral cortex of the turtle Pseudemys..I Comp. Neurol., 195 (1981) 280-304. Shatz, C. and Luskin, M.B., The relationship between the geniculocortical afferents and their cortical target cells during development of the cats primary visual cortex, .1. Veurosci~, 6 (1986) 3655-3668. Tillet, Y. and Thibault, J., Catecholamine-containing neurons in the sheep brainstem and diencephalon: immunohistochemical study with tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH) antibodies, J. Comp. NeuroL, 290 (1989) 69104. Tusa, R.J., Palmer, L.A. and Rosenquist, A.C., The retinotopic organization of area 17 in the cat, J. Comp. NeuroL, 177 (1978) 213-236. Verney, C., Molliver, M.E. and Grzanna, R., Noradrenergic innervation of the rat neocortex during development. An immunofluorescence study with antibodies raised against dopamine-beta-hydroxylase, Cr. Seances Acad. Sci, 294 (1982) 35-38. Vincent, S.R., Distributions of tyrosine hydroxylase-, dopamincbeta-hydroxylase-, and phenylethanolamine-N-methyltransferase-immunoreactive neurons in the brain of the hamster ( Mesocricetus auratus ), J. Comp. NeuroL, 268 (1988) 584-599.
ELSEVIER
Research report
Postnatal development and laminar distribution of noradrenergic fibers in cat visual cortex Yulin Liu *, Max Cynader Department of Ophthalmology, University of British Columbia, 2550 Willow Street, Vancouver, BC, Canada V5Z 3N9
Accepted 24 May 1994
Abstract Previous studies have indicated that adrenergic receptors show significant changes either in laminar distribution or in number during the critical period of kitten visual cortex development. In order to further investigate the postnatal developrnent of this neurotransmitter system, especially in relation to the critical period, we used a polyclonal antibody against dopamine-/3-hydroxylase to localize noradrenaline-containing afferents in visual cortex of kittens of various ages from birth to adulthood. In young kittens, less than 2 weeks of age, noradrenergic fibers were sparse, short and randomly oriented, and were concentrated in layer I and in deep cortical layers V and VI. By postnatal day 40, the fibers were present throughout all cortical layers and exhibited higher densities in layers I, II, III, V and VI, with a band of lower staining in layer IV. While tangential fibers predominated in layers I, V and VI, relatively straight radial fibers traversed layers II and III. After postnatal day 40, we did not find major changes in the laminar distribution of adrenergic fibers. This developmental laminar distribution pattern of adrenergic fibers resembles that of the /3-adrenergic receptors that we and others have studied in kitten visual cortex, but differs from that of a-adrenergic receptors. Keywords: Plasticity; Dopamine-/3-hydroxylase; Development; Cat; Visual cortex
1. Introduction Cortical synaptic modification due to n e u r o n a l activity occurs mainly within a short period of postnatal life and not in adult visual cortex. [11,12]. This transient 'critical p e r i o d ' has b e e n observed in the visual cortex of several species, and previous studies have shown that m a n y factors may play important roles in regulating this visual cortex plasticity [3,5,10,14]. T h e r e is evidence that cortical infusion of 6-hydroxydopamine, which destroys c a t e c h o l a m i n e terminals and depletes cortical noradrenaline, can prevent the shift o f cortical ocular d o m i n a n c e that is p r o d u c e d by m o n o c u l a r deprivation during the critical period [14], although some m o r e recent evidence points to a m o r e complex picture [3,6-8]. In previous studies, we r e p o r t e d that a - and fl-adrenergic receptors r e a c h e d their p e a k concentration in visual cortex within the critical period of visual cortex plasticity [17]. In addition, the n u m b e r and
* Corresponding author. Fax: (1) (604) 875-4663. 0165-3806/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0165-3806(94)00100-E
laminar distribution of both a - and fl-adrenergic receptors were shown to be regulated by lateral geniculate nucleus input. In o r d e r to further explore the relationship between noradrenaline and visual cortex development, we used a polyclonal antibody against dopamine-/3-hydroxylase to localize noradrenalinecontaining afferents in developing cat visual cortex and c o m p a r e d this developmental pattern with that of various classes of n o r a d r e n e r g i c receptors.
2. Materials and methods 2.1. Surgery
A total of twenty cats (see Results) of various ages were used in this study. For surgery, kittens were deeply anesthetized with halothane to effect; adult cats were anesthetized with i.v. sodium thiopental. In four animals (two kittens and two adult cats), a 1 mm thick trench of cortical tissue was aspirated by suction to a depth of 1 cm from the cortical surface (extending down to the corpus callosum) and extending from the midline to about 7 mm lateral of the midline on one side of the brain. The section was located at about A.P. 8.0 anterior and had the effect of interrupting modulatory fibers
Y. Liu, M. Cynader/ Developmental Brain Research 82 (1994) 90-94 (cholinergic, adrenergic, serotonergic and histaminergic) arising from brainstem and basal forebrain sources.
a .,
2.Z Immunocytochemistry Kittens of various ages (see Results) were used to study the normal postnatal development of dopamine-fl-hydroxylase in the developing visual cortex. Animals were deeply anesthetized with sodium pentobarbital and perfused through the aorta with 1% sodium nitrite in 0.1 M phosphate buffer (PB, pH 7.4) for 1 min followed by 4% paraformaldehyde in PB (PFA). The brains were then postfixed in 4% PFA overnight at 4°C. Blocks containing visual cortex were dissected and cut into 50 g m sections on a vibratome. After washing in PB for 1 h, the sections were incubated with a polyclonal antibody against dopamine-/3-hydroxylase (1:1000) (Anti-DBH antibody, Chemicon International Inc) and 5% normal goat serum in PB at 4°C overnight. 2.5% Triton was used to increase the penetration of the antibody within the tissue. After another series of washes in PB (6× 10 min), the samples were incubated in a second antibody (anti-rabbit IgG), followed by a series of washes in PB and incubation in avidin-biotin-HRP (1/1000, Vector) at room temperature for 45 min. Sections were then rinsed again in PB (2 x 10 min), followed by 0.05 M Tris-HCl (pH 7.6) (3 × 10 min). The immunoreaction was visualized with 0.005% 3',3'-diaminobenzidine and 0.0015% H 2 0 z. Sections were then washed in PB, dehydrated in an ascending concentration of ethanol and xylene, then coverslipped with Permount. Special care was taken to treat tissues from animals of different ages in the same way. The quality of the immunoreaction was tested by omitting the primary antibody during the process and very low immunoreactivity was found in these sections. While the DBH immunoreactive cell bodies were mostly located in the locus coeruleus of the brain stem, as has been shown frequently, the immunoreactive axons were present throughout the brain [4,9,16,21, 23,24]. The highly selective labelling observed suggests specific staining with this antibody. In addition, the results of other studies in the brains of several species have been shown that this antibody is specific in recognizing DBH [4,9,16,21,23,24]. Adjacent sections were stained with Cresyl violet and were used to establish laminar borders in the various visual cortical areas based on cytoarchitectonic criteria established by Otsuka and Hassler in adult cats and Shatz and Luskin for early postnatal development [18,20]. The electrophysiologically defined maps of Tusa which describe the relationships of regional borders and gyral patterns also contributed to defining cortical areas and layers [22]. For the lesion animals, a camera lucida system was used to draw all dopamine-/3hydroxylase immunopositive fibers within a 2 mm wide zone in the coronal plane of visual cortex of both hemispheres. Fibers throughout the cortical thickness were drawn and quantitative analysis was carried out to count the number of fibers in both the lesion and unoperated hemisphere.
3. Results
Immunoreactive fibers were found throughout the brain, with high densities in brain stem, thalamus and cortex. In the locus coeruleus of the brain stem, a number of neurons, as well as their axons were heavily stained (data not shown). 3.1. Laminar distribution and morphology of DBH-immunoreactivity in developing cat visual cortex
Kittens of various ages (postnatal day 1, 10, 20, 30, 40, 60, 90, 120 and adult) were used to study the
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c
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!'~ ~,~I ii-Ill,,..)
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Fig. 1. Photomicrographs of dopamine-fl-hydroxylase immunoreactive processes in coronal sections of developing kitten visual cortex drawn by camera lucida. Immunoreactive axons were more dense in layers I, V and VI initially, and then in older animals in layers I-III, V and VI, with lower density in layer IV. a: postnatal day 10. b: postnatal day 20. c: postnatal day 40. d: adult. Bar = 300 /~m.
developmental pattern of laminar distribution of dopamine-/3-hydroxylase in the visual cortex. The immunoreactivity was present only in axons in the visual cortex and immunoreactive axons displayed a high density varicosities throughout postnatal development (Fig. 1-3). No immunoreactive dendrites and cell bodies were found within visual cortex. In neonatal kittens, only a few immunoreactive fibers were stained. They were most concentrated in deep cortical layers and layer I. These immunoreactive fibers were confirmed as axons using morphological criteria. While the axons in layer I were mostly tangentially oriented, those found in the deep cortical layers ran in a predominantly vertical direction. At postnatal days 10 and 20, many more immunoreactive fibers were found throughout the visual cortex. These fibers were still most dense in deep cortical layers V and VI and layer I. However, many more fibers could be found in superficial layers II and III compared with postnatal day 1. At postnatal day 40, the immunoreactive fibers were most dense in the superficial layers (layers I-III) and deep cortical layers. Virtually all the immunoreactive fibers exhibited varicosities along their length (Fig. 3). After postnatal day 40, the pattern of laminar distribution of immunoreactive fibers showed little further alteration. However, the apparent density of immunoreactive fibers continued to increase until postnatal day 120. After postnatal day 120, we observed no further change either in laminar distribution or in density. That is, fibers were most dense in deep cortical layers V and VI and superficial layers I-III, with a lower band of density in layer IV. 3.2. Interruption of noradrenaline innervation to visual cortex
The effects on dopamine-3-hydroxylase immunoreactivity in cat visual cortex after lesions of the dorsal noradrenergic bundle were observed in four animals.
92
E Liu, M. Cynader / Del~elopmental Brain Research 82 (1994) 90-~)4
Two kittens were operated at 11 days of age and were perfused at postnatal day 40. Two adult cats were operated and sacrificed after a 3 month survival. The surgery was restricted to one side of the brain. We carefully counted the number of DBH positive fibers in a series of 2 mm wide fields extending through the cortical thickness in all 4 animals. Fig. 4 shows that the number of DBH immunoreactive fibers was reduced by about 80% on the lesion side (Fig. 4). However, the laminar distribution pattern of the few DBH immunoreactive fibers on the lesion side still resembled that of normal pattern of adult cat.
4. Discussion
In this study, we have used a polyclonal antibody against dopamine-/3-hydroxylase to examine the development of noradrenergic fibers in kitten visual cortex. We also examined the effect of surgical interruption of neuromodulatory pathways on the density and laminar distribution of dopamine-/3-hydroxylase immunoreactive fibers within the visual cortex. In general, noradrenergic fibers were sparse, short and randomly oriented, and were concentrated in layer I and in deep cortical layers V and VI in neonatal kittens. By 40 days of age, the fibers were present throughout all cortical layers and exhibited higher densities in layers I, II, III, V and VI, with a band of lower staining in layer IV. This developmental pattern of laminar distribution of noradrenergic fibers strongly resembles that of the development of /3-adrenergic receptors, which display a higher density of binding in deep cortical layers right after birth and then a concentration in superficial and deep cortical layers after postnatal day 40 [17]. The relatively weak DBH immunoreactivity in cortical layer IV in either neonates or during the peak of critical period is somewhat unexpected and surprised, since another group of adrenergic receptors, namely a-l- and a-2-adrenergic receptors show a strong tendency for binding in this layer and in the subplate zone at these ages [13]. In addition, this group of adrenergic receptors can be regulated by visual input. The mismatch in the distribution and laminar developmental pattern may suggest that the a-adrenergic receptors are not likely to be presynaptic receptors in visual cortex as has been described in other systems. It may be that the a-adrenergic receptors receive their inputs in a paracrine fashion primarily with long-distance diffusion from the noradrenergic fibers of nearby layers or that the relatively small number of fibers in the layer IV result in a-adrenergic receptor activation preferentially. As one of the major modulatory neurotransmitters in cerebral cortex, noradrenaline has been reported to
play an important role in regulating cortical plasticity [3,14]. The involvement of noradrenaline in regulating visual cortical plasticity has been based on evidence that cortical interruption of noradrenalinc syslcm can prevent the normal ocular dominance shift produced by monocular deprivation in kittens [1,3,8,15]. These finding are controversial but it appears likely that noradrenaline plays at least some role in cortical plasticity [3]. In this study, we showed that immunoreactive dopamine-/3-hydroxylase increased progressively after birth and reached its peak of postnatal development during the most sensitive period of visual cortex plasticity. This result provides morphological evidence to support a role for noradrenaline in regulating visual cortex development. Surgical interruption of the dorsal noradrenergic bundle depleted about 80% of dopamine-/3-hydroxyl-
Fig. 2. Photomicrographs of dopamine-fl-hydroxylase immunoreactive processes in coronal sections of adult cat visual cortex. These were most dense in superficial layers I - I l l , intermediate in layers V and VI, with the lowest density in layer IV. Bar : 300 ~tm.
Y. Liu, M. Cynader / Developmental Brain Research 82 (1994) 90-94
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Fig. 3. High magnification view showing dopamine-/3-hydroxylase immunoreactive varicosities during postnatal development. 50 g m coronal sections were taken from kitten visual cortex, a: postnatal day 1. b: postnatal day 40. c: adult. Bar = 50/xm.
ase immunoreactive fibers, yet we have shown that this manipulation has very little effect on either a- or /3-adrenergic receptor density within kitten visual cortex. The lack of effect of this major loss of noradrenergic input is somewhat surprising, but it may be that the 20% of noradrenaline fibers that remain intact provide enough input to prevent the receptor regulation that might be expected to occur. In this light, it is interesting that Daw concluded that reduction of the noradrenaline content of the visual cortex by 70 to 90% is insufficient to prevent the ocular dominance shift that occurs in the visual cortex after monocular deprivation [7,8]. The distinctive varicosities that characterized dopamine-fl-hydroxylase immunoreactive axons can be visuNormal side
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Fig. 4. Camera lucida drawing of dopamine-/3-hydroxylase immunoreactive fibers in adult animals after dorsal noradrenergic bundle lesions. Left panel: normal hemisphere, dopamine-/3-hydroxylase immunoreactive fibers were most dense in superficial and deep cortical layers. Right panel: lesioned hemisphere. About 80% depletion of dopamine-fl-hydroxylase immunoreactive fibers was observed. The heaviest concentration of fibers was still found in superficial and deep cortical layers. Bar = 300/zm.
alized throughout all the cortical layers during postnatal development. The innervation of noradrenaline to rat visual cortex has been suggested to operate at least partly through non-junctional diffusion of transmitter [19], and this pattern of innervation would affect adjacent multiple elements including neurons and glia as well as blood vessels [2,17].
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